Pancreatic cancer is the fifth leading cause of cancer death in the USA. Unfortunately, there is currently no effective screening test for asymptomatic disease and the vast majority of patients with pancreatic cancer present with advanced incurable disease. An effective method to detect early pancreatic cancers is urgently needed. This need is perhaps greatest in groups known to have an increased risk of developing pancreatic cancer. A panel of markers sensitive and specific for early stage pancreatic adenocarcinoma would greatly facilitate the early diagnosis and early detection of this disease. We propose to identify and characterize a panel of markers for early detection strategies, to test these markers in the biologic fluids of patients with pancreatic cancer compared to controls, and to determine when such markers become manifest during pancreatic neoplastic development. The two types of markers will be examined: Specific alterations in DNA methylation (Specific Aim 1), and differentially expressed proteins (Specific Aim 2) including both protein products of genes overexpressed in pancreatic carcinomas, and proteins that may be modified post-transcriptionally. For Aim 1 we will identify novel aberrantly methylated genes using MCA/RDA (representational difference analysis/methylated CpG island amplification) in a panel of ten pancreatic cancer cell lines. We will then confirm that the genes identified by MCA/RDA are aberrantly methylated in pancreatic cancer (and not normal pancreas or other tissues) by characterizing the methylation patterns of these genes in a panel of invasive pancreatic carcinomas, chronic pancreatitis and normal tissues, and determine the prevalence of aberrantly methylated genes in a panel of pancreatic precursor neoplasms, and in secondary sources including (i) the pancreatic juice and serum of patients with pancreatic cancer and controls (patients with benign neoplasms, chronic pancreatitis and no known pancreatic disease), and (ii) the pancreatic juice of patients at increased risk of pancreatic cancer undergoing EUS-based screening recruited through Project 0011. Aim 2 will encompass two strategies; (i), selecting genes overexpressed at the RNA level for protein detection studies, including immunohistochemistry, SELDI protein chip-based antibody capture (surface enhanced laser desorption ionization) and ELISA. (ii) profiling the pancreatic juice proteins of patients with pancreatic adenocarcinoma and controls using 2-dimensional gel electrophoresis, and characterizing unique proteins using MALDI-TOF mass spectroscopy.